Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca2+ channel β subunits (β1-β4) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β-GFP subunits increased the native cardiac whole-cell L-type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between β subunits, with a rank order of potency β2aαβ4 > β1b > β3. For each β subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Qmax) moved with depolarization. However, β subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (Po). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel β subunit, we cloned a new rat β2 splice variant, β2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β2a, expression of β2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L-type Ca2+ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.